Prophylactic and therapeutic monoclonal antibodies

ABSTRACT

In this application are described vaccinia monoclonal antibodies. Also provided are mixtures of antibodies of the present invention, as well as methods of using individual antibodies or mixtures thereof for the detection, prevention, and/or therapeutical treatment of vaccinia virus infections in vitro and in vivo.

This application is a continuation of application Ser. No. 09/781,124,filed Feb. 9, 2001, now U.S. Pat. No. 6,451,309 B2 which claims benefitfrom an earlier filed Provisional application Ser. No. 60/182,066 filedon Feb. 11, 2000.

INTRODUCTION

Viruses in the family Poxviridae, including vaccinia virus (VACV) andvariola virus, are characterized by a large linear double-stranded DNAgenome (130-300 kb) packaged in a relatively large virion (˜350×270 nm),and a cytoplasmic site of replication (reviewed by Moss, 1996, In“Fields Virology”, D. M. Knipe et al. Eds., vol. 3, pp 2637-2671.Lippincott-Raven, Philadelphia). Assembly of VACV virions begins withcondensation of dense granular material into membrane-wrapped particlescalled intracellular mature virions (IMV). Recent findings indiate theIMV are wrapped by a single membrane (Hollingshead et al., 1999, J.Virol. 73, 1503-1517) rather than a double membrane as previouslyreported. IMV are then enveloped in two additional membranes derivedfrom the trans Golgi to form multiple membrane-warpped particles calledintracellular enveloped virions (IEV) (Schmelz et al., 1994, J. Virol.68, 130-147). IEV are moved, possibly by actin polymerization (Cudmoreet al., 1995, Nature 378, 636-638), to the cell periphery, where theoutermost membrane fuses with the cell plasma membrane, exposing acell-associated eneveloped virion (CEV) (Blasco and Moss, 1991, J.Virol. 65, 5910-5920). CEV are released from the cell as extracellularenveloped virions (EEV), which play a role in long-range spread of thevirus (Payne, 1980, J. Gen. Virol. 50, 89-100). IMV released fromdisrupted cells and EEV are both infectious forms of VACV.

The primary therapeutic tool for the control and eradication ofinfection with VACV include a live virus vaccine to prevent disease, anda vaccinia immune globulin (VIG) to treat dissminated infections. Theexisting VIG product is derived from human donors who have beenvaccinated with the smallpox vaccine, vaccinia virus. As with all humanproducts, the existing VIG must be tested exhaustively for blood bornehuman pathogens such as human immunodeficiency virus and hepatitus B.Therefore, the existing VIG suffers from several drawbacks including thenecessity for using human volunteers, i.e. the use of a live virus as animmunogen which could cause infectious lesions that scar in healthyindividuals and severe disseminated life-threatening infection inimmunocompromised individuals. And, despite continuous screening of thedonor population to assure consistency which is very expensive, productlots can vary significantly between batches and geographic regions.

Therefore, there is a need to provide an immune globulin compositionwhich is safe and precisely defined, and which does not rely on humandonors. However, it is not known which components of the vaccinia VIGare important for protection nor how many of the ˜200 genes contained inthe vaccinia genome encode proteins that would elicit a protectiveresponse upon passive transfer of monoclonal antibodies directed to suchproteins.

SUMMARY OF THE INVENTION

This application satisfies the need mentioned above. This applicationdescribes a vaccinia immunoglobulin composition which can serve as areplacement for the presently used VIG. The vaccinia immunoglobulincomposition of the present invention is composed of one or moremonoclonal antibody against vaccinia antigens defined to be importantfor protection. To identify potential targets for poxvirus therapeutics,we generated and characterized a panel of 400 VACV-specific monoclonalantibodies (MAbs) in mice. The monoclonal antibodies were first testedfor their ability to neutralize virus and then were tested for theirability to protect mice from challenge. Two challenge models were used,one that involves dissemination of the virus (in suckling mice) and achallenge that involves a massive challenge dose (by intraperitonealinjection). To our surprise, the ability of the MAbs to inhibit plaqueformation by vaccinia virus, a standard assay of virus neutralization,did not always predict their protective efficacy. Moreover, themonoclonal antibodies differed in their ability to provide protectiondepending on the challenge model.

We found that the majority of moderately neutralizing monoclonalantibodies were directed against a 34 KDa protein later determined to beD8L which on its own did not provide protection in mice. Anothermonoclonal antibody which was neutralizing did not protect againstchallenge when given alone to mice and was directed against the proteinA27L. Neutralizing MAbs binding to the 29-KDa protein (e.g. MAb-10F5,and MAB-7D11), protected mice against intraperitoneal challenge and werefound to react with the IMV product of the L1R gene first described inWolffe, E. J. et al., 1995, Virology 211, 53-63). Nonneutralizing MAbsbinding to 23 to 28-kDA protein (e.g. MAb-1G10) protected againstchallenge with VACV (strain WR) in suckling mice. The target of MAb-1G10was the EEV product of the A33R gene (Roper et al., 1996, J. Virol. 70,3753-3762).

The LIR and A33R gene product will be called L1R and A33R, respectively.L1R is an essential myristoylated protein associated with the IMVmembrane and is thought to play a role in IMV attachment or penetration(Franke et al., 1990, J. Virol. 64, 5988-5996; Ravanello et al., 1993,J. Gen. Virol. 75, 1479-1483; Ichihashi et al., 1994, Virology 202,834-843; Ravanello and Hruby, 1994, J. Gen. Virol. 75, 1479-1483; Wolffeet al., 1995, supra). A33R is a nominally nonessentialglycosylated/palmitated protein that forms dimers and is incorporatedinto the outer membrane of EEV (Payne, 1992, Virology 187, 251-260;Roper et al., 1996, supra). A33R is thought to be involved infacilitating direct cell-to-cell spread via actin-containing microvilli(Roper et al., 1998, J. Virol. 72, 4192-4204). Homologs of L1R and A33Rare present in other Orthopoxviruses, e.g. between VACV and variola, L1Ridentity is 99.6% and A33R is 94.1% (Massung et al., 1994, Virology 201,215-240).

Therefore, it is an object of the present invention to provide acomposition of one or more monoclonal antibody directed against at leastone, preferably two or more, vaccinia virus antigens. Antigenspreferably include L1R and A33R.

It is another object of the present invention to provide monoclonalantibodies which protect against vaccinia virus infection and bind toepitopes on L1R and A33R gene products. The monoclonal antibodiesdescribed below recognize epitopes on the VACV strain Connaught L1Rsequence (Genebank #Af226617) and the Connaught strain A33R genesequence (Genebank #Af226618). L1R and A33R homologs from otherpoxviruses can be used as immunogens to produce monoclonal antibodieswhich would most likely be protective since the homologs in otherpoxviruses have high identity with the VACV proteins. Other poxvirusesinclude other Orthopoxviruses such as variola virus, monkeypox virus,cowpox virus, Parapoxviruses such as orf virus, paravaccinia virus, andunclassified poxviruses such as Tanapoxvirus, Yabapoxvirus and Molluscumcontagiosum.

It is yet another object of the present invention to provide acomposition comprising humanized monoclonal antibodies of the presentinvention for example anti-L1R antibody, or anti-A33R antibody or amixture thereof, as a vaccinia immunoglobulin replacement. The vacciniaimmunoglobulin replacement may further contain other antibodies specificfor vaccinia antigens shown to be effective for elicitingneutralizing/protective antibodies, for example H3L, D8L, B5R, A27L, andA17L. In addition, MAbs against L1R and A33R homologs from otherpoxviruses can be used alone or in combination with the vaccinia MAbs toprovide a therapeutic and prophylactic composition.

It is another object of the invention to provide for antibodies that arefunctionally equivalent to the antibodies listed above. Thesefunctionally equivalent antibodies substantially share at least onemajor functional property with an antibody listed above and hereindescribed comprising: binding specificity to L1R and A33R,immunoreactivity in vitro, protection against vaccinia challenge whenadministered prophylactically or therapeutically, competition for samebinding site on L1R and A33R. The antibodies can be of any class such asIgG, IgM, or IgA or any subclass such as IgG1, IgG2a, and othersubclasses known in the art. Further, the antibodies can be produced byany method, such as phage display, or produced in any organism or cellline, including bacteria, insect, mammal or other type of cell or cellline which produces antibodies with desired characteristics, such ashumanized antibodies. The antibodies can also be formed by combining anFab portion and a Fc region from different species.

It is another object of the present invention to provide for mixtures ofantibodies according to the present invention, as well as to methods ofusing individual antibodies, or mixtures thereof for the preventionand/or therapeutic treatment of vaccinia virus infections in vitro andin vivo, and/or for improved detection of vaccinia infections.

It is yet another object of the present invention to treat or preventvaccinia virus infection by administering a therapeutically orprophylactically effective amount of one antibody of the presentinvention or a mixture of antibodies of the present invention to asubject in need of such treatment.

It is another object of the present invention to provide passivevaccines for treating or preventing vaccinia virus infections comprisinga therapeutically or prophylactically effective amount of the antibodiesof the present invention which protect against vaccinia virus, incombination with a pharmaceutically acceptable carrier or excipient.

It is yet another object of the present invention to provide a methodfor diagnosis of vaccinia virus infection by assaying for the presenceof vaccinia in a sample using the antibodies of the present invention.

It is still another object of the present invention to provide novelimmunoprobes and test kits for detection of vaccinia virus infectioncomprising antibodies according to the present invention. Forimmunoprobes, the antibodies are directly or indirectly attached to asuitable reporter molecule, e.g., and enzyme or a radionuclide. The testkit includes a container holding one or more antibodies according to thepresent invention and instructions for using the antibodies for thepurpose of binding to vaccinia virus to form an immunological complexand detecting the formation of the immunological complex such thatpresence or absence of the immunological complex correlates withpresence or absence of vaccinia virus.

It is another object of the present invention to provide anti-idiotypicantibodies raised against one of the present monoclonal antibodies foruse as a vaccine to elicit an active anti-vaccinia response.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims, and accompanying drawings where:

FIG. 1. Passive transfer of L1R-specific MAb protects against lethalintraperitoneal challenge with VACV (strain WR). Mice were injected withthe indicated antibody and then, after 24 hrs, were challenged with VACV(strain WR). A group of 5 previously immunized mice (tail-scarified)served as positive controls. MAb-7D11 is a L1R-specific mouse MAb.MAb-1G10 is a A33R-specific mouse MAb. MAb-8E10 is a negative controlmouse MAb.

DETAILED DESCRIPTION

In the description that follows, a number of terms used in recombinantDNA, virology and immunology are extensively utilized. In order toprovide a clearer and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

The term “antibody” is art-recognized terminology and is intended toinclude molecules or active fragments of molecules that bind to knownantigens. Examples of active fragments of molecules that bind to knownantigens include Fab and F(ab′)₂ fragments. These active fragments canbe derived from an antibody of the present invention by a number oftechniques. For example, purified monoclonal antibodies can be cleavedwith an enzyme, such as pepsin, and subjected to HPLC gel filtration.The appropriate fraction containing Fab fragments can then be collectedand concentrated by membrane filtration and the like. For furtherdescription of general techniques for the isolation of active fragmentsof antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med.23:1011-1019 (1982). The term “antibody” also includes bispecific andchimeric antibodies.

The language “monoclonal antibody” is art-recognized terminology. Themonoclonal antibodies of the present invention can be prepared usingclassical cloning and cell fusion techniques. The immunogen (antigen) ofinterest, is typically administered (e.g. intraperitoneal injection) towild type or inbred mice (e.g. BALB/c) or transgenic mice which producedesired antibodies, rats, rabbits or other animal species which canproduce native or human antibodies. The immunogen can be administeredalone, or mixed with adjuvant, or expressed from a vector (VEE repliconvector, vaccinia), or as DNA, or as a fusion protein to induce an immuneresponse. Fusion proteins comprise the peptide against which an immuneresponse is desired coupled to carrier proteins, such asβ-galactosidase, glutathione S-transferase, keyhole limpet hemocyanin(KLH), and bovine serum albumin, to name a few. In these cases, thepeptides serve as haptens with the carrier proteins. After the animal isboosted, for example, two or more times, the spleen is removed andsplenocytes are extracted and fused with myeloma cells using thewell-known processes of Kohler and Milstein (Nature 256: 495-497 (1975))and Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory, New York 1988)). The resulting hybrid cells are then clonedin the conventional manner, e.g. using limiting dilution, and theresulting clones, which produce the desired monoclonal antibodies,cultured.

Monoclonal antibodies raised against vaccinia antigens L1R, A33R, H3L,D8L, B5R, A27L, and A17L are part of the present invention. Thesemonoclonal antibodies were generated from the vaccinia Connaught vaccinestrain. Other strains of vaccinia are expected to contain sequences atleast 90% identical and which will likely produce antigens capable ofeliciting protective/neutralizing antibodies. Such strains includeIHD-J, Brighton, WR, Lister, Copenhagen, Ankara, Dairen I, L-IPV,LC16M8, LC16MO, LIVP, Tian Tan, WR 65-16, Wyeth. Other homologs havingat least 90% identity exist in other poxviruses in the generaorthopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus,suipoxvirus, molluscipoxvirus and Yatapoxvirus which members includevariola major and minor virus, monkeypox virus, camelpox virus,raccoonpox virus, ectromelia virus, sealpox virus, contagious ecthymavirus, canarypox virus, juncopox virus, pigeonpox virus, turkeypoxvirus, penguinpox virus, sheepox virus, goatpox, swinepox virus,buffalopox virus, cowpox virus, rabbit fibroma virus, myxoma virus, andmolluscum contagiosum (genus Molluscipoxvirus) which is 59% identicaland 77% similar to vaccinia (Altschul, S. F. et al. 1997, Nucl. AcidsRes. 25, 3389-3402, fowlpox (genus Avipoxvirus), Yata-tumor like virus(Yatapoxvirus), among others (Fenner, Frank, Poxviruses, In “Virology”B. N. Fields et al., eds. Raves Press, Ltd. New York, 1990, pp.2113-2133). Monoclonal antibodies against homologs from these poxviruseswould likely be protective against challenge with the source ofimmunogen virus.

The term “epitope” is art-recognized. It is generally understood bythose of skill in the art to refer to the region of an antigen, such asL1R for example, that interacts with an antibody. An epitope of apeptide or protein antigen can be formed by contiguous or noncontinguousamino acid sequences of the antigen. L1R and A33R, like many proteins,contains many epitopes. The epitopes or peptides recognized by theantibodies of the present invention and conservative substitutions ofthese peptides which are still recognized by the antibody are anembodiment of the present invention. These peptides offer a convenientmethod for eluting the vaccinia antigen bound to the respective antibodyon immunoaffinity columns. For example, when an antibody whichrecognizes the epitope for L1R is used in an immunoaffinity column topurify L1R, the peptide recognized by the antibody can be added to theimmunoaffinity column to elute the L1R. Further truncation of theseepitopes may be possible since antigenic epitopes have been reported tobe represented by as few as five amino acid residues.

The antibodies described in the Examples below are characterized in thatthe antibody binds to the appropriate immunogen as measured by assayssuch as ELIA, immunoprecipitation, or immunofluorescence. Also, theL1R-specific MAbs must neutralize vaccinia virus as measured by plaqueredution neutralization test (PRNT). Any monoclonal antibody retainingthese characteristics is related to the present invention.

By further mapping of the binding site of the monoclonal antibodiesdescribed in this application other peptides useful as a vaccine or atherapeutic can be predicted. Therefore, in another aspect, thisinvention relates to a method for identifying protective antigenicepitopes the method comprising (i) reacting a monoclonal antibodydescribed in this application to different overlapping fragmentsencompassing the complete antigen, (ii) identifying a fragment to whichthe protective antibody binds, (iii) narrowing the region containingsites further by reacting the monoclonal with smaller overlappingfragments encompassing the region identified in (ii), and (iv) choosingpeptides to which the antibody binds as possible antigenic epitopes. Thepeptides can then be assayed for their ability to protect an animal fromdisease, or to reduce the severity of disease.

The epitopes or peptides on the vaccinia antigen to which the monoclonalantibodies bind can constitute all or part of an eventual active vaccinecandidate. An active vaccine or therapeutic candidate might comprisethese peptide sequences and others. These might be delivered assynthetic peptides, or as fusion proteins, alone or co-administered withcytokines and/or adjuvants or carriers safe for human use, e.g. aluminumhydroxide, to increase immunogenicity. In addition, sequences such asubiquitin can be added to increase antigen processing for more effectiveimmune responses.

The present invention also pertains to hybridomas producing antibodieswhich bind to an epitope of vaccinia antigens. The term “hybridoma” isart recognized and is understood by those of ordinary skill in the artto refer to a cell produced by the fusion of an antibody-producing celland an immortal cell, e.g. a multiple myeloma cell. This hybrid cell iscapable of producing a continuous supply of antibody. See the definitionof “monoclonal antibody” above and the Examples below for a moredetailed description of the method of fusion.

The present invention still further pertains to a method for detectingvaccinia in a sample suspected of containing vaccinia. The methodincludes contacting the sample with an antibody which binds an epitopeof a vaccinia antigen, allowing the antibody to bind to the vacciniaantigen to form an immunological complex, detecting the formation of theimmunological complex and correlating the presence or absence of theimmunological complex with the presence or absence of vaccinia antigenin the sample. The sample can be biological, environmental or a foodsample.

The language “detecting the formation of the immunological complex” isintended to include discovery of the presence or absence of vacciniaantigen in a sample. The presence or absence of vaccinia antigen can bedetected using an immunoassay. A number of immunoassays used to detectand/or quantitate antigens are well known to those of ordinary skill inthe art. See Harlow and Lane, Antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory, New York 1988 555-612). Such immunoassaysinclude antibody capture assays, antigen capture assays, andtwo-antibody sandwich assays. These assays are commonly used by those ofordinary skill in the art. In an antibody capture assay, the antigen isattached to solid support, and labeled antibody is allowed to bind.After washing, the assay is quantitated by measuring the amount ofantibody retained on the solid support. A variation of this assay is acompetitive ELISA wherein the antigen is bound to the solid support andtwo solutions containing antibodies which bind the antigen, for example,serum from a vaccinia virus vaccinee and a monoclonal antibody of thepresent invention, are allowed to compete for binding of the antigen.The amount of monoclonal bound is then measured, and a determination ismade as to whether the serum contains anti vaccinia antigen antibodies.This competitive ELISA can be used to indicate immunity to knownprotective epitopes in a vaccinee following vaccination.

In an antigen capture assay, the antibody is attached to a solidsupport, and labeled antigen is allowed to bind. The unbound proteinsare removed by washing, and the assay is quantitated by measuring theamount of antigen that is bound. In a two-antibody sandwich assay, oneantibody is bound to a solid support, and the antigen is allowed to bindto this first antibody. The assay is quantitated by measuring the amountof a labeled second antibody that can bind to the antigen.

These immunoassays typically rely on labeled antigens, antibodies, orsecondary reagents for detection. These proteins can be labeled withradioactive compounds, enzymes, biotin, or fluorochromes. Of these,radioactive labeling can be used for almost all types of assays and withmost variations. Enzyme-conjugated labels are particularly useful whenradioactivity must be avoided or when quick results are needed.Biotin-coupled reagents usually are detected with labeled streptavidin.Streptavidin binds tightly and quickly to biotin and can be labeled withradioisotopes or enzymes. Fluorochromes, although requiring expensiveequipment for their use, provide a very sensitive method of detection.Antibodies useful in these assays include monoclonal antibodies,polyclonal antibodies, and affinity purified polyclonal antibodies.Those of ordinary skill in the art will know of other suitable labelswhich may be employed in accordance with the present invention. Thebinding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known to those ofordinary skill in the art. Typical techniques are described by Kennedy,J. H., et al.,1976 (Clin. Chim. Acta 70:1-31), and Schurs, A. H. W. M.,et al. 1977 (Clin. Chim Acta 81:1-40). Coupling techniques mentioned inthe latter are the glutaraldehyde method, the periodate method, thedimaleimide method, and others, all of which are incorporated byreference herein.

The language “biological sample” is intended to include biologicalmaterial, e.g. cells, tissues, or biological fluid. By “environmentalsample” is meant a sample such as soil and water. Food samples includecanned goods, meats, and others.

Yet another aspect of the present invention is a kit for detectingvaccinia virus in a biological sample. The kit includes a containerholding one or more antibodies which binds an epitope of a vacciniaantigen and instructions for using the antibody for the purpose ofbinding to vaccinia antigen to form an immunological complex anddetecting the formation of the immunological complex such that thepresence or absence of the immunological complex correlates withpresence or absence of vaccinia virus in the sample. Examples ofcontainers include multiwell plates which allow simultaneous detectionof vaccinia virus in multiple samples.

As described in greater detail in the examples, the present inventorshave isolated monoclonal antibodies which bind to at least two differentvaccinia virus antigens, L1R and A33R, and display in vitro and/or invivo vaccinia virus protective properties. Significantly, the reactivityof the MAbs is applicable against a broad variety of different wild typeand laboratory vaccinia strains of different types.

Given these results, monoclonal antibodies according to the presentinvention are suitable both as therapeutic and prophylactic agents fortreating or preventing vaccinia infection in susceptiblevaccinia-infected subjects. Subjects include rodents such as mice orguinea pigs, birds or avian, and mammals, including humans.

In general, this will comprise administering a therapeutically orprophylactically effective amount of one or more monoclonal antibodiesof the present invention to a susceptible subject or one exhibitingvaccinia infection. Any active form of the antibody can be administered,including Fab and F(ab′)₂ fragments. Antibodies of the present inventioncan be produced in any system, including insect cells, baculovirusexpression systems, chickens, rabbits, goats, cows, or plants such astomato, potato, banana or strawberry. Methods for the production ofantibodies in these systems are known to a person with ordinary skill inthe art. Preferably, the antibodies used are compatible with therecipient species such that the immune response to the MAbs does notresult in clearance of the MAbs before virus can be controlled, and theinduced immune response to the MAbs in the subject does not induce“serum sickness” in the subject. Preferably, the MAbs administeredexhibit some secondary functions such as binding to Fc receptors of thesubject.

Treatment of individuals having vaccinia infection may comprise theadministration of a therapeutically effective amount of vacciniaantibodies of the present invention. The antibodies can be provided in akit as described below. The antibodies can be used or administered as amixture, for example in equal amounts, or individually, provided insequence, or administered all at once. In providing a patient withantibodies, or fragments thereof, capable of binding to vacciniaantigen, or an antibody capable of protecting against vaccinia virus ina recipient patient, the dosage of administered agent will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition, previous medical history, etc.

In general, it is desirable to provide the recipient with a dosage ofantibody which is in the range of from about 1 pg/kg-100 pg/kg, 100pg/kg-500 pg/kg, 500 pg/kg-1 ng/kg, 1 ng/kg-100 ng/kg, 100 ng/kg-500ng/kg, 500 ng/kg-1 ug/kg, 1 ug/kg-100 ug/kg, 100 ug/kg-500 ug/kg, 500ug/kg-1 mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100 mg/kg-500mg/kg, 500 mg/kg-1 g/kg, 1 g/kg-5 g/kg, 5 g/kg-10 g/kg (body weight ofrecipient), although a lower or higher dosage may be administered.

In a similar approach, another therapeutic use of the monoclonalantibodies of the present invention is the active immunization of apatient using an anti-idiotypic antibody raised against one of thepresent monoclonal antibodies. Immunization with an anti-idiotypic whichmimics the structure of the epitope could elicit an active anti-L1R oranti-A33R responses (Linthicum, D. S. and Farid, N. R., Anti-Idiotypes,Receptors, and Molecular Mimicry (1988), pp 1-5 and 285-300).

Likewise, active immunization can be induced by administering one ormore antigenic and/or immunogenic epitopes as a component of a subunitvaccine. Vaccination could be performed orally or parenterally inamounts sufficient to enable the recipient to generate protectiveantibodies against this biologically functional region, prophylacticallyor therapeutically. The host can be actively immunized with theantigenic/immunogenic peptide in pure form, a fragment of the peptide,or a modified form of the peptide. One or more amino acids, notcorresponding to the original protein sequence can be added to the aminoor carboxyl terminus of the original peptide, or truncated form ofpeptide. Such extra amino acids are useful for coupling the peptide toanother peptide, to a large carrier protein, or to a support. Aminoacids that are useful for these purposes include: tyrosine, lysine,glutamic acid, aspartic acid, cyteine and derivatives thereof.Alternative protein modification techniques may be used e.g.,NH₂-acetylation or COOH-terminal terminal amidation, to provideadditional means for coupling or fusing the peptide to another proteinor peptide molecule or to a support.

The antibodies capable of protecting against vaccinia virus are intendedto be provided to recipient subjects in an amount sufficient to effect areduction in the vaccinia virus infection symptoms. An amount is said tobe sufficient to “effect” the reduction of infection symptoms if thedosage, route of administration, etc. of the agent are sufficient toinfluence such a response. Responses to antibody administration can bemeasured by analysis of subject's vital signs.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

The compounds of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythese materials, or their functional derivatives, are combined inadmixture with a phamaceutically acceptable carrier vehicle. Suitablevehicles and their formulation, inclusive of other human proteins, e.g.,human serum albumin, are described, for example, in Remington'sPharmaceutical Sciences (16th ed., Osol, A. ed., Mack Easton Pa.(1980)). In order to form a pharmaceutically acceptable compositionsuitable for effective administration, such compositions will contain aneffective amount of the above-described compounds together with asuitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the compounds. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe method of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate the compounds of the present inventioninto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate)-microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

Administration of the antibodies disclosed herein may be carried out byany suitable means, including parenteral injection (such asintraperitoneal, subcutaneous, or intramuscular injection), in ovoinjection of birds, orally, or by topical application of the antibodies(typically carried in a pharmaceutical formulation) to an airwaysurface. Topical application of the antibodies to an airway surface canbe carried out by intranasal administration (e.g., by use of dropper,swab, or inhaler which deposits a pharmaceutical formulationintranasally). Topical application of the antibodies to an airwaysurface can also be carried out by inhalation administration, such as bycreating respirable particles of a pharmaceutical formulation (includingboth solid particles and liquid particles) containing the antibodies asan aerosol suspension, and then causing the subject to inhale therespirable particles. Methods and apparatus for administering respirableparticles of pharmaceutical formulations are well known, and anyconventional technique can be employed. Oral administration may be inthe form of an ingestable liquid or solid formulation.

The treatment may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of treatment may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the response, forexample, at 1-4 months for a second dose, and if needed, a subsequentdose(s) after several months. Examples of suitable treatment schedulesinclude: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii)0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient toelicit the desired responses expected to reduce disease symptoms, orreduce severity of disease.

The present invention also provides kits which are useful for carryingout the present invention. The present kits comprise a first containermeans containing the above-described antibodies. The kit also comprisesother container means containing solutions necessary or convenient forcarrying out the invention. The container means can be made of glass,plastic or foil and can be a vial, bottle, pouch, tube, bag, etc. Thekit may also contain written information, such as procedures forcarrying out the present invention or analytical information, such asthe amount of reagent contained in the first container means. Thecontainer means may be in another container means, e.g. a box or a bag,along with the written information.

The contents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLE 1

Testing L1R- and A33R-specific mouse monoclonal antibodies (MAbs) invaccinia virus (strain WR) intraperitoneal lethal challenge model.

A passive transfer experiment was performed to determine if mouse MAbsto either L1R or A33R could protect against a lethal intraperitonealchallenge with VACV (strain WR). Groups of 5 BALB/c mice (14-16 weeksold) were anesthetized, bled, and then injected (subcutaneous, behindthe base of the neck, using a 1 cc 25 G ⅝ tuberculin syringe) with 200ul of either L1R-specific MAb (MAb-7D11 ascites fluid) or A33R-specificMAb (MAb-1G10 ascities fluid) or a combination of MAb-7D11 plusMAb-1G10, or a negative control MAb (MAb-8E10, ascites fluid). Aspositive controls, 5 mice were tail-scarified with 10⁶ pfu of VACV,strain WR, approximately 3 weeks earlier. Twenty-four hours after theantibody injection, the mice were challenged with 12.5 LD₅₀ (5×10⁸ pfuin 200 ul)of VACV (strain WR).

The results are shown in FIG. 1. All of the mice injected with MAb-7D11,either alone or in combination with MAb-1G10, survived challenge. Onlyone of the five mice injected with MAb-1G10 survived. All of the micevaccinated with the negative control MAb-8E10 died. Four of the fivepositive control mice lived.

Thus, these data indicate that the L1R-specific MAb can conferprotection against a lethal challenge with VACV via the intraperitonealroute. The A33R-specific MAb failed to protect against this challenge.

EXAMPLE 2

Neonatal ICR mice were injected with the indicated antibody, as asciticfluid or protein purified antibody, and then challenged with vacciniavirus (strain IHD-J) by the subcutaneous route. The results indicatethat A33R-specific MAb-1G10 protectes against vaccinia virus (strainIHD-J) injected by the subcutaneous route whereas the L1R-specificMAb-10F5 does not. Mouse hyperimmune ascitic fluid (HMAF) alsoprotected.

Thus, these data indicate that the L1R-specific MAb can conferprotection against a lethal challenge a with VACV via theintraperitoneal route. The A33R-specific MAb failed to protect againstthis challenge. This is contrast to this MAbs capacity to protectagainst a disseminated VACV (strain IHD-J) infection in suckling mice(Table 1).

TABLE 1 Survivors/total challenged VACV challenge PFU Antibodytransferred Strain (route) (log 10) diluent HMAF Mab − 10F5 MAb − 1G1010F5 + 1G10 25 ug Ab 25 ug Ab 25 ug Ab 25 ug Ab IHD-J (s.c.) 3.9 0/139/12 0/12 10/12 9/11 25 ul AF 50 ug AB 50 ug Ab 50 ug Ab IHD-J (s.c.)3.9 0/10 8/10 0/10  9/11 (ea) combined results 0/23 17/22  0/22 19/239/11 % survival 0% 77% 0% 83% 82% IHD-J = Vaccinia virus strain IHD-Js.c. = subcutaneous PFU = plaque forming units HMAF = Vaccinia (stainConnaught) hyperimmune ascites fluid MAb − 10F5 = L1R-specific MAb MAb −1G10 = A33R-specific MAb Ab = antibody AF = ascitic fluid

What is claimed is:
 1. A composition comprising a monoclonal antibodydirected against a homolog of vaccinia L1R, a monoclonal antibodydirected against a homolog of vaccinia A33R, and at least one monoclonalantibody directed against an antigen selected from the group consistingof vaccinia H3L, D8L, B5R, A27L and A17L.
 2. The composition of claim 1,which composition further comprises at least one monoclonal antibodydirected against an antigen selected from the group consisting ofanalogs of vaccinia H3L, D8L, B5R, A27L and A17L.
 3. The composition ofclaim 1, wherein the monoclonal antibodies are reactive against at leastone poxvirus chosen from the group consisting of orthopoxvirus,parapoxvirus, avipoxvirus, capripoxvirus leporipoxvirus, suipoxvirus,molluscipoxvirus, and yatapoxvirus.
 4. The composition of claim 1wherein said composition inhibits vaccinia virus infection in a subjectin vivo.
 5. The composition of claim 4 wherein said subject is avian ormammalian.
 6. The composition of claim 1 wherein said compositionameliorates symptoms of vaccinia virus infection when said compositionis administered to a subject after infection with vaccinia virus.
 7. Thecomposition of claim 6 wherein said subject is avian or mammalian.
 8. Atherapeutic composition for ameliorating symptoms of vaccinia virusinfection comprising the composition of claim 1, and a pharmaceuticallyacceptable excipient.
 9. A passive vaccine against vaccinia virusinfection comprising the composition of claim
 1. 10. An anti-vacciniacomposition, comprising a monoclonal antibody directed against a homologof vaccinia A33R, a monoclonal antibody directed against a homolog ofvaccinia L1R, and at least one monoclonal antibody directed against anantigen selected from the group consisting of homologs of vaccinia H3L,D8L, B5R, A27L and A17L, in an amount effective for inhibiting vacciniavirus infection, and a pharmaceutically acceptable carrier.
 11. A methodof treating vaccinia virus infection comprising administering to patientin need of said treatment an effective amount of a composition accordingto claim
 1. 12. The composition according to claim 2, wherein thecomposition comprises a monoclonal antibody directed against a homologof vaccinia A33R, a monoclonal antibody directed against a homolog ofvaccinia L1R, a monoclonal antibody directed against a homolog ofvaccinia B5R, and a monoclonal antibody directed against a homolog ofvaccinia A27L.
 13. The composition of claim 1, wherein the homolog ofvaccinia L1R is at least 90% homologous to the vaccinia L1R, and thehomolog of vaccinia A33R is at least 90% homologous to the vacciniaA33R.
 14. A composition comprising a monoclonal antibody directedagainst vaccinia B5R, and a monoclonal antibody directed againstvaccinia A27L.